Inhibitor of vascular endothelial cell growth factor

ABSTRACT

The vascular endothelial cell growth factor (VEGF) inhibitors of the present invention are naturally occurring or recombinantly engineered soluble forms with or without a C-terminal transmembrane region of the receptor for VEGF, a very selective growth factor for endothelial cells. The soluble forms of the receptors will bind the growth factor with high affinity but do not result in signal transduction. These soluble forms of the receptor bind VEGF and inhibit its function.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/375,523,filed Mar. 14, 2006, which is a divisional of application Ser. No.10/101,018, filed Mar. 19, 2002, now U.S. Pat. No. 7,071,159; which is adivisional of application Ser. No. 09/232,773 filed Jan. 15, 1999, nowabandoned; which is a divisional of application Ser. No. 08/786,164filed Jan. 21, 1997, now U.S. Pat. No. 5,861,484; which is a divisionalof application Ser. No. 08/232,538 filed Apr. 21, 1994, now U.S. Pat.No. 5,712,380 which is a continuation-in-part application of applicationSer. No. 08/038,769 filed Mar. 25, 1993, now abandoned.

BACKGROUND OF THE DISCLOSURE

Recently a new class of cell-derived dimeric mitogens with selectivityfor vascular endothelial cells has been identified and designatedvascular endothelial cell growth factor (VEGF). VEGF has been purifiedfrom conditioned growth media of rat glioma cells [Conn et al., (1990),Proc. Natl. Acad. Sci. U.S.A., 87, pp 2628-2632]; and conditioned growthmedia of bovine pituitary folliculo stellate cells [Ferrara and Henzel,(1989), Biochem. Biophys. Res. Comm., 161, pp. 851-858; Gozpadorowicz etal., (1989), Proc. Natl. Acad. Sci. U.S.A., 86, pp. 7311-7315] andconditioned growth medium from human U937 cells [Connolly, D. T. et al.(1989), Science, 246, pp. 1309-1312]. VEGF is a dimer with an apparentmolecular mass of about 46 KDa with each subunit having an apparentmolecular mass of about 23 kDa. VEGF has some structural similarities toplatelet derived growth factor (PDGF), which is a mitogen for connectivetissue cells but not mitogenic for vascular endothelial cells from largevessels.

The membrane-bound tyrosine kinase receptor, known as FLT, was shown tobe a VEGF receptor [DeVries, C. et al., (1992), Science, 255, pp.989-991]. The FLT receptor specifically binds VEGF which inducesmitogenesis. Another form of the VEGF receptor, designated KDR, is alsoknown to bind VEGF and induce mitogenesis. The partial cDNA sequence andnearly full length protein sequence of KDR is known as well [Terman, B.I. et al., (1991) Oncogene 6, pp. 1677-1683; Terman, B. I. et al.,(1992) Biochem. Biophys. Res. Comm. 187, pp, 1579-1586].

Persistent angiogenesis may cause or exacerbate certain diseases such aspsoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabeticretinopathy and neovascular glaucoma. An inhibitor of VEGF activitywould be useful as a treatment for such diseases and other VEGF-inducedpathological angiogenesis and vascular permeability conditions, such astumor vascularization.

SUMMARY OF THE DISCLOSURE

A naturally-occurring FLT messenger RNA (mRNA) was identified and clonedfrom vascular endothelial cells. This mRNA is shown to encode most ofthe extracellular, or soluble, portion of the VEGF receptor, FLT.Soluble receptor molecules including forms containing a C-terminaltransmembrane region are also recombinantly engineered for this andother VEGF receptors. These soluble receptors, comprising truncated andmodified forms are expressed in recombinant host cells and have VEGFbinding properties. The soluble receptor proteins are useful asinhibitors of VEGF activity since they will bind available VEGFpreventing it from activating its functional receptors on vascularendothelial cells and could form non-functional heterodimers withfull-length membrane anchored VEGF receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—A schematic diagram of full length VEGF receptors (FLT and KDR),the soluble VEGF receptors (sVEGF-RI and sVEGF-RII) and the solublereceptors containing the C-terminal transmembrane region (VEGF-RTMI andVEGF-RTMII) are shown with the protein domains of each.

FIG. 2—The DNA sequence of the sVEGF-RI soluble VEGF receptor/VEGFinhibitor is shown.

FIG. 3—The amino acid sequence of the sVEGF-RI soluble VEGFreceptor/VEGF inhibitor is shown.

FIG. 4—Demonstration that recombinant host cells express sVEGF-RI isshown by the formation of high molecular weight complexes of sVEGF-RIand [¹²⁵I]VEGF and separated by size exclusion chromatography.

FIG. 5—A 12.5% polyacrylamide electrophoretic gel is shown whichdemonstrates the high degree of purity obtained for sVEGF-RI.

FIG. 6—Cross-linked products of sVEGF-RI and [¹²⁵I]VEGF are shown atabout 145 kDa, and at about 245 kDa.

FIGS. 7A and 7B—Analysis of VEGF binding to sVEGF-RI (A) andcorresponding Scatchard plot (B).

FIG. 8—Inhibition of [¹²⁵I]VEGF binding to HUVECs by sVEGF-RI isdemonstrated.

FIG. 9—Inhibition of VEGF-mediated mitogenesis on HUVECs is shown usingsVEGF-RI.

FIG. 10—The nucleotide sequence encoding sVEGF-RII is shown.

FIG. 11—The amino acid sequence for sVEGF-RII is shown.

FIG. 12—The nucleotide sequence encoding VEGF-RTMII is shown.

FIG. 13—The amino acid sequence fors V-EGF-RTMII is shown.

FIG. 14—The nucleotide sequence encoding sVEGF-RTMI is shown.

FIG. 15—The amino acid sequence for sVEGF-RTMI is shown.

FIG. 16—A diagram of pmFLT is shown.

FIG. 17—A diagram of pKDRA is shown.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present invention relates to cDNA encoding a soluble VEGF receptorprotein (sVEGF-R) which is isolated from VEGF receptor producing cellsor is recombinantly engineered from VEGF receptor-encoding DNA. sVEGF-R,as used herein, refers to a protein which can specifically bind to avascular endothelial cell growth factor without stimulating mitogenesisof vascular endothelial cells.

The amino acid sequence of FLT is known, [Shibuya, M. et al., (1990),Oncogene, 5, pp. 519-524] and corresponds to the full lengthcell-associated VEGF tyrosine kinase receptor. Other VEGF receptors areknown to exist. Other known VEGF receptors include, but are not limitedto KDR [Terman (1991), supra., and Terman (1992), supra.]. Mammaliancells capable of producing FLT, KDR and other VEGF receptors include,but are not limited to, vascular endothelial cells. Mammalian cell lineswhich produce FLT or KDR and other VEGF receptors include, but are notlimited to, human endothelial cells. The preferred cells for the presentinvention include human umbilical vein endothelial cells (HUVEC).

Other cells and cell lines may also be suitable for use to isolatesVEGF-R cDNA. Selection of suitable cells may be done by screening forsVEGF-R binding activity on cell surfaces, in cell extracts orconditioned medium or by screening for gene expression by PCR orhybridization. Methods for detecting soluble receptor activity are wellknown in the art [Duan, D-S. R. et al., (1991) J. Biol. Chem., 266, pp.413-418] and measure the binding of labelled VEGF. Cells which possessVEGF binding activity in this assay may be suitable for the isolation ofsVEGF-R cDNA.

Full length FLT producing cells such as human IC cells (American TypeCulture Collection, ATCC CRL 1730) [Hoshi, H. and McKeehan, W. L., Proc.Natl. Acad. Sci. U.S.A., (1984) 81, pp. 6413-6417] are grown accordingto the recommended culture conditions of the ATCC. Full length FLT, andKDR VEGF receptors as well as extracellular region (sVEGF-RI andsVEGF-RII) and extracellular region plus transmembrane region forms(VEGF-RTMI and VEGF-RTMII) are shown in FIG. 1. The full length receptorhas an extracellular ligand binding region composed of about sevenimmunoglobulin-like domains, a membrane spanning sequence (transmembranedomain) and intracellular tyrosine kinase domains. The inhibitory formsof this receptor, which are the subject of the present invention, arealso shown in FIG. 1 and lack the intracellular kinase domains, and forsome inhibitors, the transmembrane sequence and the C-terminal mostIg-like extracellular domain.

Any of a variety of procedures may be used to molecularly clone sVEGF-RcDNA. These methods include, but are not limited to, direct functionalexpression of the sVEGF-R gene following the construction of ansVEGF-R-containing cDNA library in an appropriate expression vectorsystem.

Another method is to screen a sVEGF-R-containing cDNA libraryconstructed in a bacteriophage or plasmid shuttle vector with a labelledoligonucleotide probe designed from the predicted amino acid sequence ofsVEGF-R. The preferred method consists of screening a sVEGF-R-containingcDNA library constructed in a bacteriophage or plasmid shuttle vectorwith a partial cDNA encoding at least part of the full length FLTprotein. This partial cDNA is obtained by the specific PCR amplificationof sVEGF-R DNA fragments through the design of oligonucleotide primersfrom the known sequence of the full length FLT-encoding DNA.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cells or celltypes, may be useful for isolating sVEGF-R-encoding DNA. Other types oflibraries include, but are not limited to, cDNA libraries derived fromother cells or cell lines other than HUVECs and genomic DNA libraries.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have sVEGF-Ractivity. The selection of cells or cell lines for use in preparing acDNA library to isolate sVEGF-R cDNA may be done by first measuringsecreted sVEGF-R activity using the VEGF binding assay described fullyherein.

Preparation of cDNA Libraries can be Performed by Standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1982).

It is also readily apparent to those skilled in the art that DNAencoding sVEGF-R may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in Maniatis, T., Fritsch,E. F., Sambrook, J. in Molecular Cloning: A Laboratory Manuel (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982).

Another means of obtaining sVEGF-R molecules is to recombinantlyengineer them from DNA encoding the partial or complete amino acidsequence of a VEGF receptor. Examples of other VEGF receptors include,but are not limited to, KDR. Using recombinant DNA techniques, DNAmolecules are constructed which encode at least a portion of the VEGFreceptor capable of binding VEGF without stimulating mitogenesis.Standard recombinant DNA techniques are used such as those found inManiatis, et al., supra.

Using one of the preferred methods of the present invention, cDNA clonesencoding sVEGF-R are isolated in a two-stage approach employingpolymerase chain reaction (PCR) based technology and cDNA libraryscreening. In the first stage, DNA oligonucleotides derived from theextracellular domain sequence information from the known full lengthFLT, KDR or other VEGF receptor is used to design degenerateoligonucleotide primers for the amplification of sVEGF-R-specific DNAfragments. In the second stage, these fragments are cloned to serve asprobes for the isolation of complete sVEGF-R cDNA from a commerciallyavailable lambda gt10 cDNA library (Clontech) derived from HUVEC cells(ATCC CRL 1730).

These PCR derived products were used as hybridization probes forscreening a lambda gt10 cDNA library derived from HUVECs (Clontech).Plating and plaque lifts of the library were performed by standardmethods (T. Maniatis, E. F. Fritsch, J. Sambrook, Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982). The probes were random-primed labelled with ³²P-dCTP tohigh specific activity and a separate screening of the library (1×10⁶plaques per screen) was conducted with each probe. The probes were addedto hybridization buffer (50% formamide, 5×Denhardts, 6×SSC (1×SSC 0.15 MNaCl, 0.015 M Na₃citrate·2H₂O, pH 7.0), 0.1% SDS, 100 mg/ml salmon spermDNA) at 1×10⁶ cpm/ml.

Four positively hybridizing phage were detected using the flt-specificprobe. These positively hybridizing phage were observed to be less thanfull length flt.

Two fit cDNA clones of about 2.0 kb and 2.7 kb in length were subclonedinto pGEM vectors (Promega) and bi-directionally sequenced in theirentirety by the chain termination method (Sanger et al., (1977) P.N.A.S.USA, 74, pp. 5463-5467,) and shown to contain a single open readingframe of about 569 amino acids. Sequence analysis demonstrated that aportion of the 5′ flt coding region was missing from these clones. Theremainder of the 5′ end was cloned using PCR and combined with the DNAof the clones lacking the 5′ end to yield a single open reading frameencoding about 687 amino acids.

The sequence for the cDNA encoding flt-derived sVEGF-RI is shown inTable 1, and was identified in clones 7 and 11. The deduced amino acidsequence of sVEGF-RI from the cloned cDNA is shown in Table 2.Inspection of the deduced amino acid sequence reveals the presence of asingle, large open reading frame of 687 amino acids. By comparison withamino acid sequence of the full length FLT VEGF receptor, 31 amino acidsare encoded at the C-terminal end of the cDNA which are different fromthose of FLT.

Using another of the preferred methods of the present invention, DNAencoding sVEGF-R is constructed from a DNA sequence encoding a VEGFreceptor. For purposes of illustration, DNA encoding the VEGF receptorknown as KDR was utilized. Using the receptor DNA sequence, a DNAmolecule is constructed which encodes the extracellular domain of thereceptor, or the VEGF binding domain only and is denoted sVEGF-RII.Restriction endonuclease cleavage sites are identified within thereceptor DNA and can be utilized directly to excise theextracellular-encoding portion. In addition, PCR techniques as describedabove may be utilized to produce the desired portion of DNA. It isreadily apparent to those skilled in the art that other techniques,which are standard in the art, may be utilized to produce sVEGF-Rmolecules in a manner analagous to those described above. Suchtechniques are found, for example, in Maniatis et al., supra.

Additional truncated forms of the VEGF receptor are constructed whichcontain the transmembrane region. Retention of the transmembrane mayfacilitate orientation of the inhibitor molecule at the target cellsurface. Examples of transmembrane region containing inhibitor moleculesinclude but are not limited to those shown in FIG. 1. VEGF-RTMI andVEGF-RTMII, as shown in FIG. 1, are FLT-related and KDR-related,respectively, transmembrane region containing receptor inhibitors.Construction of transmembrane region containing molecules, such asVEGF-RTMI and VEGF-RTMII, is done by standard techniques known in theart including but not limited to utilizing convenient restrictionendonuclease cleavage sites or PCR techniques as described herein. It isreadily understood by those skilled in the art that various forms of theinhibitors of a VEGF receptor, as disclosed herein, containing only theextracellular region or containing, in addition, the transmembraneregion may be constructed which have substantially the same activity.

The cloned sVEGF-R cDNA obtained through the methods described above maybe recombinantly expressed by molecular cloning into an expressionvector containing a suitable promoter and other appropriatetranscription regulatory elements, and transferred into prokaryotic oreukaryotic host cells to produce recombinant sVEGF-R. Techniques forsuch manipulations are fully described in Maniatis, T, et al., supra,and are well known in the art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria, bluegreenalgae, fungal cells, yeast cells, plant cells, insect cells and animalcells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal or bacteria-insect cells. Anappropriately constructed expression vector should contain: an origin ofreplication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and active promoters. A promoter isdefined as a DNA sequence that directs RNA polymerase to bind to DNA andinitiate RNA synthesis. A strong promoter is one which causes mRNAs tobe initiated at high frequency. Expression vectors may include, but arenot limited to, cloning vectors, modified cloning vectors, specificallydesigned plasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant sVEGF-R in mammalian cells. Commercially available mammalianexpression vectors which may be suitable for recombinant sVEGF-Rexpression, include but are not limited to, pMC1neo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2)(ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), andgZD35 (ATCC 37565).

DNA encoding sVEGF-R may also be cloned into an expression vector forexpression in a recombinant host cell. Recombinant host cells may beprokaryotic or eukaryotic, including but not limited to bacteria, yeast,mammalian cells including but not limited to cell lines of human,bovine, porcine, monkey and rodent origin, and insect cells includingbut not limited to drosophila, moth, mosquito and armyworm derived celllines. Cell lines derived from mammalian species which may be suitableand which are commercially available, include but are not limited to,CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH3T3 (ATCC CRL 1658), HeLa (ATCC CCL2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL171). Insect cell lines which may be suitable and are commerciallyavailable include but are not limited to 3M-S (ATCC CRL 8851) moth (ATCCCCL 80) mosquito (ATCC CCL 194 and 195; ATCC CRL 1660 and 1591) andarmyworm (Sf9 ATCC CRL 1711).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, liposome or protoplast fusion, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce sVEGF-R protein.Identification of sVEGF-R expressing host cell clones may be done byseveral means, including but not limited to immunological reactivitywith anti-sVEGF-R antibodies, binding to radiolabelled VEGF, and thepresence of host cell-secreted sVEGF-R activity.

Expression of sVEGF-R DNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being preferred.

Levels of sVEGF-R protein produced by host cells may be quantitated byimmunoaffinity and/or ligand affinity techniques. sVEGF-R-specificaffinity beads or sVEGF-R-specific antibodies are used to isolate³⁵S-methionine labelled or unlabelled sVEGF-R protein. Labelled sVEGF-Rprotein is analyzed by SDS-PAGE. Unlabelled sVEGF-R protein is detectedby Western blotting, ELI SA or RIA assays employing sVEGF-R specificantibodies, or by ligand blotting with labelled VEGF.

Following expression of sVEGF-R in a recombinant host cell, sVEGF-Rprotein may be recovered to provide sVEGF-R in active form, capable ofbinding VEGF without stimulating mitogenesis. Several sVEGF-Rpurification procedures are available and suitable for use. sVEGF-R maybe purified from cell lysates and extracts, or from conditioned culturemedium, by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography, reversedphase chromatography, heparin sepharose chromatography, VEGF ligandaffinity chromatography, and hydrophobic interaction chromatography.

In addition, recombinant sVEGF-R can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for full length sVEGF-R, or polypeptidefragments of sVEGF-R.

Identification of sVEGF-RI—In an attempt to clone the VEGF receptor cDNA(flt) a HUVEC lgt10 cDNA library was screened with a DNA probe derivedfrom the extracellular domain of the membrane bound or full length formof this receptor as shown in FIG. 1. Four incomplete clones, all lackingvarious lengths of 5′ coding sequence, were isolated from screening atotal of 1×10⁶ plaques. Two of these isolates represent partial clonesthat were identical to full length flt, one of which contained thecomplete 3′ coding region of the form described by Shibuya et al.,supra. The other two clones were identical to full length flt up to basepair number 2219 (Table 1 and FIG. 2) where they then diverged from fulllength flt. These clones (clone 7 and 11) coded for an additional unique31 amino acids before the open reading frame is terminated by a TAAcodon (Table 2 and FIG. 3).

Clone 7 and 11 coded for a protein with a predicted molecular mass ofabout 75 kDa containing 12 putative N-linked glycosylation sites. Thisversion of the receptor was missing the transmembrane and intracellularkinase domains and thus coded for a natural soluble form of the VEGFreceptor (sVEGF-Ri). Further, the protein molecule predicted by sVEGF-RIhas only the first six Ig-like domains, missing the one closest to thetransmembrane sequence (FIG. 1). The 31 amino acids at the C-terminalend of sVEGF-RI contain two cysteine residues, but does not resemble anIg domain.

Expression of sVEGF-RI in S89 cells—To analyze the binding andbiological properties of this form of the receptor, the protein wasexpressed using a baculovirus expression system. Clone 7 was missingabout 350 base pairs of coding sequence at the 5′ end. This region wascloned by PCR using the primers described above and in Example 1. Aclone containing the complete coding region of sVEGF-RI was constructedby combining the 5 PCR fragment with sVEGF-RI clone 7 which overlappedat a SacI site. The 5′ EcoRI site was then changed to a BamHI site andthe full length sVEGF-RI was cloned into pBluebac III (Invitrogen) as aBamHI/BamHI fragment. A recombinant baculovirus P-3 stock containing thesVEGF-RI gene 3′ in relation to the polyhedrin promoter was thenprepared as described herein.

Culture media from small scale infections were tested for the ability toform high molecular weight complexes with [¹²⁵I]VEGF. The labeled ligandand culture media from the baculovirus infected cells were combined andincubated. The reactions were then analyzed by size exclusionchromatography. When the wild-type infected culture medium was mixedwith the radioactive ligand (FIG. 4) a single radioactive peak wasobserved. However, when the sVEGF-RI infected culture medium was used, ahigh molecular weight complex was formed, as evident by the appearanceof a second peak in this reaction eluting near the void volume of thecolumn. This experiment showed that the natural soluble form of the FLTVEGF receptor, sVEGF-RI, forms a high molecular weight complex withVEGF.

The recombinantly produced sVEGF-R is purified from the recombinant hostcell extracts or cell culture fluid using heparin-sepharose columnchromatography which specifically binds the sVEGF-R protein. Theheparin-sepharose bound VEGF-R column is washed using a suitable buffercontaining between 0.1M and 0.6M NaCl which removes contaminatingproteins without significant loss of sVEGF-R. The sVEGF-R is eluted fromthe heparin-sepharose column using a suitable buffer containing about MNaCl, yielding substantially purified sVEGF-R.

Binding of the sVEGF-RI to VEGF—The binding of ¹²⁵I-labelled VEGF tosVEGF-RI was characterized by crosslinking, and by complex formationwith sVEGF-RI absorbed to 96 well plates.

The crosslinked products are shown in FIG. 6. The sVEGF-RI wascross-linked to [¹²⁵I]VEGF (lane 1); in the presence of unlabelled VEGF(lane 2) and unlabelled bFGF (lane 3). Two high molecular weight bands(about 145 kDa and 245 kDa) were formed in the sVEGF-RI and [¹²⁵I]VEGFcontaining reaction, and in the sVEGF-RI and [¹²⁵I]VEGF plus an excessof unlabelled bFGF reaction. The two high molecular weight bands werenot present when sVEGF-RI was incubated with [¹²⁵I]VEGF plus an excessof unlabelled VEGF, demonstrating the specificity of sVEGF-RI for VEGF,and the ability of sVEGF-RI to form a dimer. The 145 kDa band ispresumably a crosslinked complex containing one receptor molecule (about100 kDa) and a VEGF dimer (about 46 kDa). As shown in FIG. 6 complexescontaining two receptor molecules (about 245 kDA) were also observed.This suggests that each VEGF dimer can bind one or two receptormolecules and that the soluble form of the VEGF receptor may undergoligand-induced dimerization.

The affinity of sVEGF-RI for VEGF was evaluated by absorbing sVEGF-RI tothe surface of a 96 well plate, followed by blocking the nonspecificsites with 0.5% gelatin. Variable amounts of labeled ligand were addedto each well. These results demonstrate that sVEGF-RI binds VEGF withhigh affinity with an apparent K_(d) of about 20 pM (FIG. 7). Since thesoluble form of the receptor is missing the Ig domain closest to thetransmembrane spanning region, this domain is not required for ligandbinding.

The sVEGF-RI is shown to inhibit binding of VEGF to HUVECs by incubatingcultured HUVECs with [¹²⁵I]VEGF and various amounts of sVEGF-RI.Following incubation, the cells are washed to remove unbound [¹²⁵I]VEGF.The cells are then solubilized and the amount of cell-associated [¹²⁵I]is determined by gamma counter, which demonstrates the amount of[¹²⁵I]VEGF which was capable of binding to the cellular VEGF receptor inthe presence of sVEGF-RI. Using this method, it is demonstrated thatsVEGF-RI was capable of inhibiting VEGF binding to HUVECs VEGF receptor(see FIG. 8).

Since sVEGF-RI was able to inhibit VEGF binding to cell receptors, itwas then determined that sVEGF-RI could inhibit VEGF inducedmitogenesis. Cells are preincubated with sVEGF-RI and then incubatedwith VEGF in the presence of [³H]thymidine. Following incubation, theamount of cellular DNA-incorporated [³H]thymidine is measured whichindicates whether VEGF has induced mitogenesis and caused [³H]thymidineto be incorporated into cellular DNA. The presence of sVEGF-RI inhibitsthe ability of VEGF to stimulate mitogenesis as shown in FIG. 9.

The inhibitor of the present invention can be used for the inhibition ofVEGF activity. The inhibitor can be used either topically orintravascularly. For topical applications the formulation would beapplied directly at a rate of about 10 ng to about 1 mg/cm²/day. Forintravaneous applications, the inhibitor is used at a rate of about 1 mgto about 10 mg/kg/day of body weight. For internal use, the formulationmay be released directly into the region to be treated either fromimplanted slow release polymeric material or from slow release pumps orrepeated injections. The release rate in either case is about 100 ng toabout 100 mg/day/cm³.

For non-topical application the VEGF inhibitor is administered incombination with pharmaceutically acceptable carriers or diluents suchas phosphate buffer, saline, phosphate buffered saline, Ringer'ssolution, and the like, in a pharmaceutical composition, according tostandard pharmaceutical practice. For topical application, variouspharmaceutical formulations are useful for the administration of theactive compound of this invention. Such formulations include, but arenot limited to, the following: ointments such as hydrophilic petrolatumor polyethylene glycol ointment; pastes which may contain gums such asxanthan gum; solutions such as alcoholic or aqueous solutions; gels suchas aluminum hydroxide or sodium alginate gels; albumins such as human oranimal albumins; collagens such as human or animal collagens; cellulosessuch as alkyl celluloses, hydroxy alkyl celluloses and alkylhydroxyalkylcelluloses, for example methylcellulose, hydroxyethyl cellulose,carboxymethyl cellulose, hydroxypropyl methylcellulose, andhydroxypropyl cellulose; polyoxamers such as Pluronic® Polyolsexemplified by Pluronic® F-127; tetronics such as tetronic 1508; andalginates such as sodium alginate.

The following examples are provided as illustrative of the presentinvention without, however, limiting the same thereto.

Example 1

Cloning flt-related sVEGF-RI—A 580 base pair DNA probe for flt wasobtained by PCR of the HUVEC phage library using the primers 5′GCACCTTGGTTGTGGCTGAC 3′ (SEQ. ID. No.: 1) and 5′TGGAATTCGTGCTGCTTCCTGGTCC 3′ (SEQ. ID. No.: 2). The resulting DNAfragment was cloned into pGEM3Z as a XbaI/EcoRI fragment. The probe wasprepared by the random priming method [Feinberg, A. P. and Vogelstein,B., (1983) Anal. Biochem., 132, pp. 6-13] using the megaprime kit(Amersham) at a specific activity of 1×10⁷ cpm/ng. The HUVEC cDNAlibrary was plated at a density of 5×10⁴ plaques/150 cm plate then about1×10⁶ plaques were screened by hybridization as previously described[Maniatis, T. et al., supra]. Briefly, following prehybridization at 42°C. for 2 hours in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.1% SDS,100 mg/ml salmon sperm DNA (hybridization buffer) the filters werehybridized with the probe for 16 hours at 42° C. in hybridizationbuffer. The filters were washed one time for 15 min at room temperaturein 2×SSC then three times at 55° C. in 0.1×SSC. Four positive plaqueswere identified and rescreened two additional times to obtainhomogeneous isolates. Inserts were cloned into pGEM3Z for DNA sequenceanalysis. Two of these clones were identified which contained less thanthe full length flt coding region. DNA sequence analysis showed thatthese clones lacked the 5′ coding region of flt. The DNA sequence isshown in Table 1 and FIG. 2, and the deduced amino acid sequence isshown in Table 2 and FIG. 3. The 5′ end of flt was cloned by PCR usingthe primers 5′ GGAATTCCGCGCTCACCATGGTCAGC 3′ (SEQ.ID.NO.:3) and 5′TTTGAATTCACCCGGCAGGGAATGACG 3′ (SEQ.ID.NO.:4). The PCR fragmentgenerated with this set of primers was cloned into fit clone 7 as anEcoRI/SacI fragment.

TABLE 1 (SEQ. ID. NO.: 5) GCGGACACTCCTCTCGGCTCCTCCCCGGCAGCGGCGGCGGCTCGGAGCGGGCTCCGGGGCTCGGGTGCAGCGGCCAGCGGGCCTGGCGGCGAGGATTACCCGGGGAAGTGGTTGTCTCCTGGCTGGAGCCGCGAGACGGCCGCTCAGGGCGCGGGGCCGGCGGCGGCGAACGAGAGGACGGACTCTGGCGGCCGGGTCGTTGGCCGGGGGAGCGCGGGCACCGGGCGAGCAGGCCGCGTCGCGCTCACC ATG GTC AGC TAC TGG GAC ACC GGGGTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTC ACA GGA TCT AGT TCA GGTTCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG CAC ATC ATG CAAGCA GGC CAG ACA CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA TGG TCTTTG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT GCCTGT GGA AGA AAT GGC AAA CAA TTC TGC AGT ACT TTA ACC TTG AAC ACA GCT CAAGCA AAC CAC ACT GGC TTC TAC AGC TGC AAA TAT CTA GCT GTA CCT ACT TCA AAGAAG AAG GAA ACA GAA TCT GCA ATC TAT ATA TTT ATT AGT GAT ACA GGT AGA CCTTTC GTA GAG ATG TAC AGT GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGGGAG CTC GTC ATT CCC TGC CGG GTT ACG TCA CCT AAC ATC ACT GTT ACT TTA AAAAAG TTT CCA CTT GAC ACT TTG ATC CCT GAT GGA AAA CGC ATA ATC TGG GAC AGTAGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGG CTT CTG ACCTGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CAT CGACAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TTACTT AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACGAGA GTT CAA ATG ACC TGG AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTAAGG CGA CGAATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTTCTTACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT TGTCGT GTAAGG AGT GGA CCA TCA TTC AAA TCT GTT AAC ACC TCA GTGCAT ATA TAT GAT AAAGCA TTC ATC ACT GTG AAA CAT CGA AAA CAGCAG GTG CTT GAA ACC GTA GCT GGCAAG CGG TCT TAC CGG CTC TCTATG AAA GTG AAG GCA TTT CCC TCG CCG GAA GTTGTA TGG TTA AAAGAT GGG TTA CCT GCG ACT GAG AAA TCT GCT CGC TAT TTG ACTCGT GGC TAC TCG TTA ATT ATC AAG GAC GTA ACT GAA GAG GAT GCA GGG AAT TATACA ATC TTG CTG AGC ATA AAA CAG TCA AAT GTG TTT AAA AAC CTC ACT GCC ACTCTA ATT GTC AAT GTG AAA CCC CAG ATT TAC GAA AAG GCC GTG TCA TCG TTT CCAGAC CCG GCT CTC TAC CCA CTG GGC AGC AGA CAA ATC CTG ACT TGT ACC GCA TATGGT ATC CCT CAA CCT ACA ATC AAG TGG TTC TGG CAC CCC TGT AAC CAT AAT CATTCC GAA GCA AGG TGT GAC TTT TGT TCC AAT AAT GAA GAG TCC TTT ATC CTG GATGCT GAC AGC AAC ATG GGA AAC AGA ATT GAG AGC ATC ACT CAG CGC ATG GCA ATAATA GAA GGA AAG AAT AAG ATG GCT AGC ACC TTG GTT GTG GCT GAC TCT AGA ATTTCT GGA ATC TAC ATT TGC ATA GCT TCC AAT AAA GTT GGG ACT GTG GGA AGA AACATA AGC TTT TAT ATC ACA GAT GTG CCA AAT GGG TTT CAT GTT AAC TTG GAA AAAATG CCG ACG GAA GGA GAG GAC CTG AAA CTG TCT TGC ACA GTT AAC AAG TTC TTATAC AGA GAC GTT ACT TGG ATT TTA CTG CGG ACA GTT AAT AAC AGA ACA ATG CACTAC AGT ATT AGC AAG CAA AAA ATG GCC ATC ACT AAG GAG CAC TCC ATC ACT CTTAAT CTT ACC ATC ATG AAT GTT TCC CTG CAA GAT TCA GGC ACC TAT GCC TGC AGAGCC AGG AAT GTA TAC ACA GGG GAA GAA ATC CTC CAG AAG AAA GAA ATT ACA ATCAGA GGT GAG CAC TGC AAC AAA AAG GCT GTT TTC TCT CGG ATC TCC AAA TTT AAAAGC ACA AGG AAT GAT TGT ACC ACACAAAGTAATGTAAAACATTAAAGGACTCATTAAAAAGTAACAGTTGTCTCATATCATCTTGATTTATTGTCAGTGTTGCTAACTTTCAGGCTCGGAGGAGATGCTCCTCCCAAAATGAGTTCGGAGATGATAGCAGTAATAATGAGACCCCCGGGCTCCAGCTCTGGGCCCCCCATTCAGGCCGAGGGGGCTGCTCCGGGGGGCCGACTTGGTGCACGTTTGGATTTGGAGGATCCCTGCACTGCCTTCTCTGTGTTTGTTGCTCTTGCTGTTTTCTCCTGCCTGATAAACAACAACTTGGGATGATCCTTTCCATTTTGATGCCAACCTCTTTTTATTTTTAAG CGGCGCCCTATAGT

TABLE 2 (SEQ. ID. NO.: 6) Met Val Ser Tyr Trp Asp Thr Gly Val Leu LeuCys Ala Leu Leu Ser Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys LeuLys Asp Pro Gln Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly GlnThr Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro GlnMet Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala Cys Gly ArgAsn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn HisThr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys GluThr Glu Ser Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val GluMet Tyr Ser Gln Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu ValIle Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe ProLeu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys GlyPhe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu AlaThr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr AsnThr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg GlyHis Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val GlnMet Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg ArgIle Asp Gln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile AspLys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly ProSer Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile ThrVal Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser TyrArg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val Trp Leu LysAsp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr SerLeu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu LeuSer Ile Lys Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Leu Ile Val AsnVal Lys Pro Gln Ile Tyr Glu Lys Ala Val Ser Ser Phe Pro Asp Pro Ala LeuTyr Pro Leu Gly Ser Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro GlnPro Thr Ile Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala ArgCys Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser AsnMet Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile Glu Gly LysAsn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg Ile Ser Gly Ile TyrIle Cys Ile Ala Ser Asn Lys Val Gly Thr Val Gly Arg Asn Ile Ser Phe TyrIle Thr Asp Val Pro Asn Gly Phe His Val Asn Leu Glu Lys Met Pro Thr GluGly Glu Asp Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp ValThr Trp Ile Leu Leu Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile SerLys Gln Lys Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr IleMet Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn ValTyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg Gly Glu HisCys Asn Lys Lys Ala Val Phe Ser Arg Ile Ser Lys Phe Lys Ser Thr Arg AsnAsp Cys Thr Thr Gln Ser Asn Val Lys His***

Example 2

Expression of sVEGF-RI in Sf9 insect cells—The full length sequenceencoding sVEGF-RI was cloned as an EcoRI/BamHI f fragment into pGEM3Z.The EcoRI site was then modified to a BamHI site and cloned intopBlueBac III 3′ of the polyhedrin promoter (psFLTblue). This plasmid wastransfected into Sf9 armyworm cells using liposomes. After 48 hours themedium from the transfected cells which contains recombinant polyhedrinvirus particles, was harvested. Dilutions (10³-10⁴ fold) of the viruswere prepared and plaque purified in soft agar containing 150 mg/ml5-bromo-4-chloro-3-indolyl-β-D-galactoside. Recombinant plaques wereidentified by blue color and used to infect Sf9 cells (5×10⁵ cells/well)in 12 well plates. Medium (100 ml) from polyhedrin minus infections wasused to prepare P-2 viral stocks by infecting 2.5×10⁶ cells in a T-25flask. Large scale high titer P-3 viral stocks were then prepared byinfecting Sf9 cells (500 ml at 2×10⁶ cells/ml) with 5 ml of the P-2stock then incubating at 27° C. for 5-6 days and the medium washarvested by centrifugation. Protein expression was accomplished byinfecting cells at a density of 2-2.5×10⁶ cells/ml with a multiplicityof infection of 5-10. Twenty four hours after infection the cells werechanged to a serum free medium (SF900II, Gibco BRL), incubated for anadditional 48 hours and the medium was collected. This conditionedmedium contains the recombinantly expressed sVEGF-RI protein.

Example 3

Iodination of VEGF and PIGF—¹²⁵I-labeled human recombinant VEGF wasprepared by the chloramine T method (Hunter, W. M. and Greenwood, F. C.,(1962) Nature (London), 194, pp. 495-496). Briefly, 1 mg of VEGF in 30%acetonitrile/0.1% trifluoroacetic acid was adjusted to pH 7.1 by theaddition of ⅓ volume of 0.4 M sodium phosphate buffer, pH 7.1. Freshlydissolved chloramine T (4 ml of a 2 mg/ml stock in 0.1 M sodiumphosphate buffer, pH 7.1) was added to the VEGF solution and reacted for45 seconds at room temperature (total volume of 150 ml). The reactionwas stopped by the addition of 50 ml of 10 mM KI and 50 ml of 2 mg/mlmeta bisufite. The labeled ligand was separated from the free ¹²⁵I bygel filtration on a 0.7×15 cm Sephadex G-25 column equilibrated in PBSwith 1 mg/ml gelatin. Fractions were counted in a Packard g counter,aliquoted and stored at −70° C. VEGF was labeled to a specific activityof 5×10⁵ to 1×10⁶ cpm/ng. Recombinant human PlGF was iodinated by thechloramine-T method as described herein, to specific activity betweenapproximately 3×10⁵−9×10⁵ cpm/ng. After iodination, PlGF was stored at4° C. in PBS containing 1 mg/ml gelatin.

Gel Filtration Chromatography—Receptor-ligand complex was formed byincubating 10 ml of ¹²⁵I-labeled VEGF (10⁵ cpm) with 100 ml of eitherwild-type or baculovirus sVEGF-RI-containing, infected Sf9 cell culturemedium overnight at room temperature. The reaction products wereseparated on a Sephacryl S200 gel filtration column (0.7×25 cm)equilibrated in PBS, 1 mg/ml gelatin, at a flow rate of 15 ml/hr.Fractions (0.75 ml) were collected and analyzed in a g counter.Receptor-ligand complexes pass quickly through the column while the freelabelled VEGF passes through more slowly. The results of this experimentshown in FIG. 4 demonstrate the formation of a high molecular weightcomplex between labelled VEGF and sVEGF-RI protein. This shows thatsVEGF-RI binds VEGF.

Crosslinking—Purified sVEGF-RI (1-10 ng) was added to 25 ml of bindingbuffer (Dulbecco's Modified Eagle's medium (DM), 25 mM HEPES, pH 7.5,0.3% gelatin), and 1×10⁵ cpm of [¹²⁵I]-VEGF was added (FIG. 6, lane 1)with either 200 ng of unlabelled VEGF (lane 2) or bFGF (lane 3), thenincubated 2 to 16 hours at room temperature.Bis(sulfosuccinimidyl)suberate (Pierce) crosslinker was added to a finalconcentration of 1 mM. The reaction was stopped after 15 min by theaddition of boiling SDS PAGE sample buffer. The crosslinked productswere separated by SDS PAGE on a 7.5% acrylamide gel and analyzed eitherby autoradiography or a phosphoimager. The results are shown in FIG. 6and demonstrate that sVEGF-RI binds labelled VEGF by the appearance oftwo bands of about 145 kDa and 245 kDa. The 145 kDa band consists of onesVEGF-RI molecule and one VEGF molecule (Monomer, M.). The 245 kDa bandapparently consists of two sVEGF-RI molecules and one VEGF dimer (D).Free VEGF ligand (L) dimers migrated at about 45 kDA.

Purified Ex-KDR and sFLT were each allowed to bind either [¹²⁵I]VEGF or[¹²⁵I]PlGF at 25° C. for 1 hr in a final volume of 25 μl in bindingbuffer (10 mM Hepes, pH 7.4, 0.01% BSA, 100 mM NaCl) with or without anexcess of the appropriate unlabeled ligand. Competition binding wasaccomplished by incubation in the presence of various concentrations ofunlabeled VEGF (0.1-400 nM). The reactions were then crosslinked with 1mM BS³ at 25° C. for 15 min followed by the addition of boiling Laemmlisample buffer (10). The crosslinked products were analyzed by SDS/7.5%PAGE and the complexes were visualized using a PhosphoImager (MolecularDynamics, Sunnyvale, Calif.). In the competition crosslinkingexperiments the amount of radioactivity contained in theEx-KDR/[¹²⁵I]VEGF complex as well as the uncomplexed [¹²⁵I]VEGF werequantified using the PhosphoImager.

Binding assay—The binding of sVEGF-RI to VEGF was analyzed using a 96well plate assay as described by Duan, D-S. R. et al. supra. Briefly,sVEGF-RI, 50 to 200 ml partially purified by Mono Q chromatography(Pharmacia), was diluted to 10 ml in 25 mM TRIS, pH 7.4, 100 mM NaCl, 20mM NH₄HCO₃. Aliquots (100 ml) were absorbed to the surface of a 96 wellplate for 18 hours at 4° C., the plates were then washed twice withblocking buffer (DME, 25 mM HEPES, pH 7.5, 0.5% gelatin) and thenonspecific sites were blocked in the same buffer for 6 hours at 4° C.The plate was then washed twice in binding buffer. Various amounts of[¹²⁵I]VEGF were added to the wells in a final volume of 100 ml/well andincubated for 2 hours at room temperature. The wells were washed threetimes with 100 ml of binding buffer, the bound protein was solubilizedwith 100 ml of 1% SDS, 0.5% BSA and counted in a g counter. The results,shown in FIG. 7, were analyzed by the method of Scatchard [Scatchard,G., (1949) Ann. N.Y. Acad. Sci., 51, pp. 660-672]. The analysisdemonstrates that sVEGF-RI retains high affinity binding for VEGF with aK_(d) value of about 20 pM. This clearly demonstrates that sVEGF-RI,lacking the transmembrane region and adjacent Ig-like domain, binds VEGFwith high affinity and that these regions are not required for VEGFbinding.

Purified Ex-KDR and sFLT were each allowed to bind either [₁₂₅I]VEGF or[¹²⁵I]PlGF at 25° C. for 1 hour in a final volume of 25 μl in bindingbuffer (10 mM Hepes, pH 7.4, 0.01% BSA, 100 mM NaCl) with or without anexcess of the appropriate unlabeled ligand. Competition binding wasaccomplished by incubation in the presence of various concentrations ofunlabeled VEGF (0.1-400 nM). The reactions were then crosslinked with 1mM BS³ at 25° C. for 15 min followed by the addition of boiling Laemmlisample buffer. The crosslinked products were analyzed by SDS/7.5% PAGEand the complexes were visualized using a PhosphoImager (MolecularDynamics, Sunnyvale, Calif.). In the competition crosslinkingexperiments the amount of radioactivity contained in theEx-KDR/[¹²⁵I]VEGF complex as well as the uncomplexed [¹²⁵I]VEGF werequantified using the PhosphoImager.

To determine if sFLT and Ex-KDR bind VEGF and PlGF with high affinity,purified sFLT and Ex-KDR were each incubated with either [¹²⁵I]VEGF or[¹²⁵I]PlGF, covalently crosslinked and high molecular mass complexeswere resolved by SDS/PAGE. sFLT formed high molecular mass complexeswith both VEGF and PlGF whereas Ex-KDR formed complexes with VEGF butnot with PlGF. The positions of the monomer (one VEGF dimer bound to onereceptor molecule) and dimer (one VEGF dimer bound to two receptormolecules) were as expected. These radiolabeled complexes were competedby an excess of the same unlabeled VEGF or PlGF and thus are specific.PlGF was able to compete for VEGF binding to the sFLT receptor and VEGFcompetes for PlGF binding to this receptor. PlGF was not able to competefor [¹²⁵]VEGF binding to Ex-KDR.

The affinity of VEGF for Ex-KDR was determined by a crosslinkingcompetition binding assay since the Ex-KDR receptor binds poorly to 96well plates. A constant amount of [¹²⁵I]VEGF was bound to Ex-KDR in thepresence of increasing concentrations of unlabeled VEGF. Theconcentration of unlabeled VEGF required to displace 50% of the total[¹²⁵I]VEGF binding is approximately 1 nM, which is similar to theapparent K_(d) for the membrane form of KDR.

Competition Between PlGF and VEGF for Binding to sFLT

Competitive binding of VEGF and PlGF to sFLT was analyzed by the 96 wellplate binding assay. A constant amount of either [¹²⁵I]VEGF or[¹²⁵I]PlGF was bound to immobilized sFLT in the presence of increasingamounts of either unlabeled VEGF or PlGF. In comparison, 50% of thebinding of [¹²⁵I]PlGF to sFLT was displaced by only 10 pM of VEGF.Approximately 110 pM of unlabeled PlGF displaced 50% of [¹²⁵I]PlGFbinding to sFLT in agreement with saturation binding experiments.However, an approximately 5-fold higher concentration of PIGF (˜550 pM)was required to displace 50% of the [¹²⁵I]VEGF binding to sFLT. Thesedata indicate that VEGF and PlGF compete for the same site on sFLT atwhich VEGF binds with ˜4-fold higher affinity than PlGF. Crosslinkingcompetition experiments with sFLT gave similar results.

Here we show that VEGF binds to the extracellular domains of both FLTand KDR with high affinity. PlGF, however, only binds to theextracellular domain of FLT with high affinity and does not bind to theequivalent extracellular region of KDR. VEGF is able to competeefficiently for PlGF binding to sFLT whereas PlGF competes lessefficiently for VEGF binding. These binding data demonstrate that VEGFcomplexes with sFLT somewhat tighter than does PlGF. Competitive bindinginfers that the VEGF and PlGF sites on sFLT are probably eitheroverlapping or identical. Thus, sFLT will inhibit both PlGF and VEGFfunction.

Example 4

Inhibition of VEGF binding by sVEGF-RI—The ability of sVEGF-RI toinhibit VEGF binding to HUVECs was tested. HUVECs were plated at 50,000cells/well in 24 well plates precoated with gelatin, and allowed to growto confluence. A constant amount of [¹²⁵I]VEGF (100,000 cpm) was mixedwith various amounts of partially purified sVEGF-RI in binding buffer,in a total volume of 200 μl and preincubated at room temperature for 1hour. Samples were added to the cells and incubated for 4 hours at 4° C.with shaking. The medium was then aspirated and the cells were washedthree times with binding buffer. The bound radioactivity was solubilizedwith 50 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 1% NP40, 1% BSA and counted ina y counter.

The results are shown in FIG. 8. At the highest concentration ofsVEGF-RI, VEGF binding to HUVECs was reduced by 70%. It may, however, bedifficult to completely inhibit binding to the cellular membrane boundreceptor since one molecule of sVEGF-R bound to a VEGF dimer may be ableto bind to cell associated receptor to form an inactive(sVEGF-RI)-VEGF-(membrane spanning VEGF receptor) complex.

Example 5 Inhibition of VEGF Mediated Mitogenesis by sVEGF-RI

Mitogenic inhibition—Since sVEGF-RI was able to inhibit VEGF binding toendothelial cells, it was then determined that the soluble receptorcould inhibit VEGF induced mitogenesis in HUVECs. HUVECs were plated ingelatin coated 96 well plates at a density of 4000 cells/well in 100 mlof DME supplemented with 10% heat inactivated fetal calf serum plusantibiotics (penicillin G, 100 units/ml; streptomycin sulfate, 100mg/ml). After 16 hours the medium was changed and test samples wereadded, cells were preincubated with a variable amount of purifiedsVEGF-RI for 15 minutes at 37° C. before growth factor (10 ng/ml) wasadded. The cells were incubated for 24 hours then [methyl-3H]thymidine(0.8 mCi/well; 20 Ci/mmol: 1Ci=37 GBq, final specific activity of 0.8mCi/nmole) was added followed by incubated for an additional 72 hours at37° C. under 5% CO₂. The cells were then washed twice with Hank'sbalanced salt solution adjusted to pH 7.5 with 25 mM Hepes, 0.1% BSA.The cells were then lysed, the DNA was solubilized with 0.2 M Na₂CO₃,0.1 M NaOH, and [³H]thymidine incorporation was quantified byscintillation counting. The results are shown in FIG. 9. sVEGF-RI wasable to completely inhibit VEGF induced [³H]thymidine incorporation inHUVECs.

Example 6

Purification of baculovirus expressed sVEGF-RI from Sf9 cells—Culturemedium from Sf9 cells infected with a baculovirus construct designed toexpress sVEGF-RI (Example 2) was chromatographed through a heparinSepharose CL-6B (Pharmacia) column (0.7×4 cm). The column was washedwith 5 volumes of 10 mM Na-phosphate buffer, pH 6.2, 0.1 M NaCl,followed by 6 ml of 10 mM Na-phosphate buffer, pH 6.2, 0.6 M NaCl. ThesVEGF-RI was eluted with 10 m Na-phosphate buffer, pH 6.2, 1.0 M NaCl.Polyacrylamide gel electrophoresis was performed which demonstratedgreater than 90% purity (as judged by coomassie blue staining) of therecombinantly produced sVEGF-R (FIG. 5). The identity of the protein wasconfirmed by N-terminal protein sequence analysis. The actual N-terminus(Ser Lys Leu . . . ) of the recombinant protein differs by two aminoacids from that predicted by Shibuya et al., supra. (Ser-Ser-Ser . . .). The peptidase cleavage site in sVEGF-RI produced in Sf9 cells wasbetween residues gly-26 and ser-27.

Example 7

Construction of KDR-related sVEGF-R—Soluble forms of KDR (a known VEGFreceptor) [Terman, B. I. et al., (1991) Oncogene 6, pp. 1677-1683;Terman, B. I. et al., (1992) Biochem. Biophys. Res. Comm. 187, pp.1579-1586] may exist naturally but have not yet been identified. Asoluble form of KDR is recombinantly constructed by modifying its codingsequence by PCR using the primers 1) 5′TTTTGGATCCCTGCAGACAGATCTACGTTTGAGAACC 3′ (SEQ. ID. NO.: 7) and 2) 5′TTTTGGATCCTTAACGCTCTAGGACTGTGAGC 3′ (SEQ. ID. NO.: 8), and pKDRA (theXhoI/EcoRI fragment coding for the extracellular and transmembranedomain of KDR cloned into the EcoRI site of pGEM 7Z obtained fromPromega) as a template (FIG. 17). This generated a translation stopcodon after amino acid residue number 663 of KDR which corresponds tothe extracellular domain of full length KDR. This modified fragment isthen used to replace the Pstl/BamHl fragment of pKDRA generating atruncated form of the KDR gene (FIG. 10) which codes for a solublereceptor denoted sVEGF-RII (FIG. 11). The Xhol site at base pair number257 is then changed to a BamHl site by standard cloning techniques.Another truncated form of the KDR receptor is created with primer 1shown above, and primer 3) 5′ TTTTGGATCCAACGGTCCCTAGGATGATGAC 3′, (SEQ.ID. NO.: 9) (FIG. 12). This form of KDR, denoted VEGF-RTMII, istruncated at the C-terminal side of the transmembrane domain andtherefore retains the transmembrane region (FIG. 13). A similar form ofthe FLT receptor is generated by PCR using the primers 4) 5′AGCACCTTGGTTGTGGCTGACTC 3′ (SEQ. ID. NO.: 10) and 5) 5′TTTTGGATCCTTAGATAAGGAGGGTTAATAGG 3′ (SEQ. ID. NO.: 11) and plasmid pmFLT(full length flt cloned into the EcoRI site of pGEM3Z obtained fromPromega) as a template (FIG. 16). The 780 base pair PCR fragment canthen be cloned together with the EcoRl/Xbal fragment from pmFLT toproduce an EcoRl/BAMHl fragment (FIG. 14) encoding a truncated form ofFLT (denoted VEGF-RTMI) which retains the transmembrane domain but lacksthe cytoplasmic domain (FIG. 15). The EcoRl site at the 5′ end of thegene is then modified to a BamHl site. The resulting truncated forms ofKDR and FLT are then cloned into pBluebaclll (Stratagene) for expressionin Sf9 insect cells. Characterization of these constructed truncatedforms of VEGF receptors is accomplished by the techniques used tocharacterize sVEGF-RI as in Examples 2, 3, 4, 5, and 6.

Example 8 Identification and Partial Purification of a Soluble VEGFBinding Protein

A mRNA encoding a soluble version of Flt was expressed in HUVECs. Therecombinant sFlt protein, when expressed in Sf9 insect cells (BVsFlt),was found to bind tightly to heparin Sepharose. To determine if sFltprotein was expressed by HUVECs, conditioned medium from cultured HUVECswas filtered through a 0.22 μm membrane and passed over a heparinsepharose column. The heparin column was eluted with a step gradient andfractions were tested for binding to [¹²⁵I] VEGF by covalentcrosslinking. VEGF binding activity eluted at similar NaClconcentrations as the BVsFlt protein and was found in the 0.6-1.2 M NaClstep fraction. An equal volume of EndoUV medium (endothelial cell growthmedium) not conditioned was chromatographed and had no VEGF bindingactivity in the 0.6-1.2 M NaCl fraction. The VEGF binding activity fromHUVECs when crosslinked to labeled VEGF formed complexes which migrateslower on SDS/PAGE than VEGF complexes formed with BVsFlt. VEGF bindingfractions were pooled and further separated by cation exchangechromatography with a linear NaCl gradient. Again, VEGF binding activityfrom the endothelial cell conditioned medium elutes at a similarposition as BVsFlt.

The chromatography data shows that the partially purified HUVEC VEGFbinding protein behaves similar to BVsFlt. To determine if this VEGFbinding protein is related to Flt, antibodies against peptides based onthe N-terminus and third immunoglobulin-like domain in the extracellularregion of Flt were prepared. Fractions from the mono S column thatproduced high molecular weight complexes when covalently crosslinked to[¹²⁵I] VEGF were analyzed by Western blot analysis. These data show thata 116 kDa protein band which co-elutes with VEGF binding activity wasdetected by both antibodies, thus the binding activity isolated fromhuman endothelial cells is a soluble form of Flt.

1. A soluble VEGF inhibitor in substantially pure form whichspecifically binds VEGF and inhibits cellular VEGF receptor activity. 2.The soluble VEGF inhibitor according to claim 1 wherein the soluble VEGFreceptor is selected from the group consisting of sVEGF-RI, sVEGF-RII,sVEGF-RTMI and sVEGF-RTMII.
 3. The soluble VEGF inhibitor of claim 2corresponding to sVEGF-RI comprising the amino acid sequence: (SEQ. ID.NO.: 6) Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu SerCys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro Glu LeuSer Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr Leu His Leu GlnCys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro Glu Met Val Ser Lys GluSer Glu Arg Leu Ser Ile Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln PheCys Ser Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr SerCys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala IleTyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu IlePro Glu Ile Ile His Met Thr Glu Gly Avg Glu Leu Val Ile Pro Cys Arg ValThr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu IlePro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser AsnAla Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly HisLeu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp ValGln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val LeuAsn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser TyrPro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser AsnSer His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn LysAsp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser ValAsn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val Lys His Arg LysGln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met LysVal Lys Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro AlaThr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile Lys AspVal Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln SerAsn Val Phe Lys Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln IleTyr Glu Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly SerArg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile Lys TrpPhe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys Asp Phe Cys SerAsn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser Asn Met Gly Asn Arg IleGlu Ser Ile Thr Gln Arg Met Ala Ile Ile Glu Gly Lys Asn Lys Met Ala SerThr Leu Val Val Ala Asp Ser Arg Ile Ser Gly Ile Tyr Ile Cys Ile Ala SerAsn Lys Val Gly Thr Val Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val ProAsn Gly Phe His Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu LysLeu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu LeuArg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys Met AlaIle Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met Asn Val Ser LeuGln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn Val Tyr Thr Gly Glu GluIle Leu Gln Lys Lys Glu Ile Thr Ile Arg Gly Glu His Cys Asn Lys Lys AlaVal Phe Ser Arg Ile Ser Lys Phe Lys Ser Thr Arg Asn Asp Cys Thr Thr GlnSer Asn Val Lys His.


4. The soluble VEGF inhibitor of claim 2 corresponding to sVEGF-RIcomprising the amino acid sequence: (SEQ. ID. NO.: 12) Ser Lys Leu LysAsp Pro Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln ThrLeu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro Glu MetVal Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala Cys Gly Arg AsnGly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His ThrGly Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu ThrGlu Ser Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu MetTyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val IlePro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro LeuAsp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly PheIle Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala ThrVal Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn ThrIle Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly HisThr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gln MetThr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg IleAsp Gln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp LysMet Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro SerPhe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr ValLys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser Tyr ArgLeu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys AspGly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser LeuIle Ile Lys Asp Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu SerIle Lys Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Leu Ile Val Asn ValLys Pro Gln Ile Tyr Glu Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu TyrPro Leu Gly Ser Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln ProThr Ile Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg CysAsp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser Asn MetGly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile Glu Gly Lys AsnLys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg Ile Ser Gly Ile Tyr IleCys Ile Ala Ser Asn Lys Val Gly Thr Val Gly Arg Asn Ile Ser Phe Tyr IleThr Asp Val Pro Asn Gly Phe His Val Asn Leu Glu Lys Met Pro Thr Glu GlyGlu Asp Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val ThrTrp Ile Leu Leu Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser LysGln Lys Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile MetAsn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn Val TyrThr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg Gly Glu His CysAsn Lys Lys Ala Val Phe Ser Arg Ile Ser Lys Phe Lys Ser Thr Arg Asn AspCys Thr Thr Gln Ser Asn Val Lys His.


5. The soluble VEGF inhibitor of claim 2 corresponding to sVEGF-RIIcomprising the amino acid sequence: (SEQ. ID. NO.: 13)MQSKVLLAVALWLCVETRAASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIAVNKNQFALIEGKNKTVSTLVIQAANVSALYKCEAVNKVGRGERVISFHVTRGPELTLQPDMQPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLER.


6. The soluble VEGF inhibitor of claim 2 corresponding to sVEGF-RTMIcomprising the amino acid sequence: (SEQ. ID. NO.: 14)MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNYYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQLTWFKNNHKIQQEPGIILGPGSSTLFIERVTEEDEGVYHCKATNQKGSVESSAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLL I.


7. The soluble VEGF inhibitor of claim 2 corresponding to sVEGF-RTMIIcomprising the amino acid sequence: (SEQ. ID. NO.: 15)MQSKVLLAVALWLCVETRAASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAEGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIAVNKNQFALIEGKNKTVSTLVIQAANYSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEVSCTASGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYCQACSVLGCAKVFAFFIIEGAQEKTNLEIIILVGTT VIAMFFWLLLVIILGTV.


8. An expression vector comprising a promoter, and a DNA sequenceencoding a soluble VEGF inhibitor for expression in recombinant hostcells wherein the soluble VEGF inhibitor is selected from the groupconsisting of sVEGF-RI, sVEGF-RII, sVEGF-RTMI and sVEGF-RTMII.
 9. Theexpression vector of claim 8 wherein the DNA encoding the sVEGF-RIcomprises the nucleotide sequence: (SEQ. ID. NO.: 5)GCGGACACTCCTCTCGGCTCCTCCCCGGCAGCGGCGGCGGCTCGGAGCGGGCTCCGGGGCTCGGGTGCAGCGGCCAGCGGGCCTGGCGGCGAGGATTACCCGGGGAAGTGGTTGTCTCCTGGCTGGAGCCGCGAGACGGGCGCTCAGGGCGCGGGGCCGGCGGCGGCGAACGAGAGGACGGACTCTGGCGGCCGGGTCGTTGGCCGGGGGAGCGCGGGCACCGGGCGAGCAGGCCGCGTCGCGCTCACC ATG GTC AGC TAC TGG GAC ACC GGGGTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTC ACA GGA TCT AGT TCA GGTTCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG CAC ATC ATG CAAGCA GGC CAG ACA CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA TGG TCTTTG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT GCCTGT GGA AGA AAT GGC AAA CAA TTC TGC AGT ACT TTA ACC TTG AAC ACA GCT CAAGCA AAC CAC ACT GGC TTC TAC AGC TGC AAA TAT CTA GCT GTA CCT ACT TCA AAGAAG AAG GAA ACA GAA TCT GCA ATC TAT ATA TTT ATT AGT GAT ACA GGT AGA CCTTTC GTA GAG ATG TAC AGT GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGGGAG CTC GTC ATT CCC TGC CGG GTT ACG TCA CCT AAC ATC ACT GTT ACT TTA AAAAAG TTT CCA CTT GAC ACT TTG ATC CCT GAT GGA AAA CGC ATA ATC TGG GAC AGTAGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGG CTT CTG ACCTGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CAT CGACAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TTACTT AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACGAGA GTT CAA ATG ACC TGG AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTAAGG CGA CGA ATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTTACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGGAGT GGA CCA TCA TTC AAA TCT GTT AAC ACC TCA GTG CAT ATA TAT GAT AAA GCATTC ATC ACT GTG AAA CAT CGA AAA CAG CAG GTG CTT GAA ACC GTA GCT GGC AAGCGG TCT TAC CGG CTC TCT ATG AAA GTG AAG GCA TTT CCC TCG CCG GAA GTT GTATGG TTA AAA GAT GGG TTA CCT GCG ACT GAG AAA TCT GCT CGC TAT TTG ACT CGTGGC TAC TCG TTA ATT ATC AAG GAC GTA ACT GAA GAG GAT GCA GGG AAT TAT ACAATC TTG CTG AGC ATA AAA CAG TCA AAT GTG TTT AAA AAC CTC ACT GCC ACT CTAATT GTC AAT GTG AAA CCC CAG ATT TAC GAA AAG GCC GTG TCA TCG TTT CCA GACCCG GCT CTC TAC CCA CTG GGC AGC AGA CAA ATC CTG ACT TGT ACC GCA TAT GGTATC CCT CAA CCT ACA ATC AAG TGG TTC TGG CAC CCC TGT AAC CAT AAT CAT TCCGAA GCA AGG TGT GAC TTT TGT TCC AAT AAT GAA GAG TCC TTT ATC CTG GAT GCTGAC AGC AAC ATG GGA AAC AGA ATT GAG AGC ATC ACT CAG CGC ATG GCA ATA ATAGAA GGA AAG AAT AAG ATG GCT AGC ACC TTG GTT GTG GCT GAC TCT AGA ATT TCTGGA ATC TAC ATT TGC ATA GCT TCC AAT AAA GTT GGG ACT GTG GGA AGA AAC ATAAGC TTT TAT ATC ACA GAT GTG CCA AAT GGG TTT CAT GTT AAC TTG GAA AAA ATGCCG ACG GAA GGA GAG GAC CTG AAA CTG TCT TGC ACA GTT AAC AAG TTC TTA TACAGA GAC GTT ACT TGG ATT TTA CTG CGG ACA GTT AAT AAC AGA ACA ATG CAC TACAGT ATT AGC AAG CAA AAA ATG GCC ATC ACT AAG GAG CAC TCC ATC ACT CTT AATCTT ACC ATC ATG AAT GTT TCC CTG CAA GAT TCA GGC ACC TAT GCC TGC AGA GCCAGG AAT GTA TAC ACA GGG GAA GAA ATC CTC CAG AAG AAA GAA ATT ACA ATC AGAGGT GAG CAC TGC AAC AAA AAG GCT GTT TTC TCT CGG ATC TCC AAA TTT AAA AGCACA AGG AAT GAT TGT ACC ACA CAA AGT AAT GTA AAA CAT TAAAGGACTCATTAAAAAGTAACAGTTGTGTCATATCATCTTGATTTATTGTCACTGTTGCTAACTTTCAGGCTCGGAGGAGATGCTCCTCCCAAAATGAGTTCGGAGATGATAGCAGTAATAATGAGACCCCCGGGCTCCAGCTCTGGGCCCCCCATTCAGGCCGAGGGGGCTGCTCCGGGGGGCCGACTTGGTGCACGTTTGGATTTGGAGGATCCCTGCACTGCCTTCTCTGTGTTTGTTGCTCTTGCTGTTTTCTCCTGCCTGATAAACAACAACTTGGGATGATCCTTTCCATTTTGATGCCAACCTCTTTTTATTTTTAAGCGGCGCCCTATAGT.


10. The expression vector of claim 8 wherein the DNA encoding thesVEGF-RII comprises the nucleotide sequence: (SEQ. ID. NO.: 16)GGTGTGGTCGCTGCGTTTCCTCTGCCTGCGCCGGGCATCACTTGCGCGCCGCAGAAAGTCCGTCTGGCAGCCTGGATATCCTCTCCTACCGGCACCCGCAGACGCCCCTGCAGCCGCGGTCGGCGCCCGGGCTCCCTAGCCCTGTGCGCTCAACTGTCCTGCGCTGCGGGGTGCCGCGAGTTCCACCTCCGCGCCTCCTTCTCTAGACAGGCGCTGGGAGAAAGAACCGGCTCCCGAGTTCCGGCATTTCGCCCGGCTCGAGGTGCAGGATGCAGAGCAAGGTGCTGCTGGCCGTCGCCCTGTGGCTCTGCGTGGAGACCCGGGCCGCCTCTGTGGGTTTGCCTAGTGTTTCTCTTGATCTGCCCAGGCTCAGCATACAAAAAGACATACTTACAATTAAGGCTAATACAACTCTTCAAATTACTTGCAGGGGACAGAGGGACTTGGACTGGCTTTGGCCCAATAATCAGAGTGGCAGTGAGCAAAGGGTGGAGGTGACTGAGTGCAGCGATGGCCTCTTCTGTAAGACACTCACAATTCCAAAAGTGATCGGAAATGACACTGGAGCCTACAAGTGCTTCTACCGGGAAACTGACTTGGCCTCGGTCATTTATGTCTATGTTCAAGATTACAGATCTCCATTTATTGCTTCTGTTAGTGACCAACATGGAGTCGTGTACATTACTGAGAACAAAAACAAAACTGTGGTGATTCCATGTCTCGGGTCCATTTCAAATCTCAACGTGTCACTTTGTGCAAGATACCCAGAAAAGAGATTTGTTCCTGATGGTAACAGAATTTCCTGGGACAGCAAGAAGGGCTTTACTATTCCCAGCTACATGATCAGCTATGCTGGCATGGTCTTCTGTGAAGCAAAAATTAATGATGAAAGTTACCAGTCTATTATGTACATAGTTGTCGTTGTAGGGTATAGGATTTATGATGTGGTTCTGAGTCCGTCTCATGGAATTGAACTATCTGTTGGAGAAAAGCTTGTCTTAAATTGTACAGCAAGAACTGAACTAAATGTGGGGATTGACTTCAACTGGGAATACCCTTCTTCGAAGCATCAGCATAAGAAACTTGTAAACCGAGACCTAAAAACCCAGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGTGTAACCCGGAGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTGGGCTGATGACCAAGAAGAACAGCACATTTGTCAGGGTCCATGAAAAACCTTTTGTTGCTTTTGGAAGTGGCATGGAATCTCTGGTGGAAGCCACGGTGGGGGAGCGTGTCAGAATCCCTGCGAAGTACCTTGGTTACCCACCCCCAGAAATAAAATGGTATAAAAATGGAATACCCCTTGAGTCCAATCACACAATTAAAGCGGGGCATGTACTGACGATTATGGAAGTGAGTGAAAGAGACACAGGAAATTACACTGTCATCCTTACCAATCCCATTTCAAAGGAGAAGCAGAGCCATGTGGTCTCTCTGGTTGTGTATGTCCCACCCCAGATTGGTGAGAAATCTCTAATCTCTCCTGTGGATTCCTACCAGTACGGCACCACTCAAACGCTGACATGTACGGTCTATGCCATTCCTCCCCCGCATCACATCCACTGGTATTGGCAGTTGGAGGAAGAGTGCGCCAACGAGCCCAGCCAAGCTGTCTCAGTGACAAACCCATACCCTTGTGAAGAATGGAGAAGTGTGGAGGACTTCCAGGGAGGAAATAAAATTGCCGTTAATAAAAATCAATTTGCTCTAATTGAAGGAAAAAACAAAACTGTAAGTACCCTTGTTATCCAAGCGGCAAATGTGTCAGCTTTGTACAAATGTGAAGCGGTCAACAAAGTCGGGAGAGGAGAGAGGGTGATCTCCTTCCACGTGACCAGGGGTCCTGAAATTACTTTGCAACCTGACATGCAGCCCACTGAGCAGGAGAGCGTGTCTTTGTGGTGCACTGCAGACAGATCTACGTTTGAGAACCTCACATGGTACAAGCTTGGCCCACAGCCTCTGCCAATCCATGTGGGAGAGTTGCCCACACCTGTTTGCAAGAACTTGGATACTCTTTGGAAATTGAATGCCACCATGTTCTCTAATAGCACAAATGACATTTTGATCATGGAGCTTAAGAATGCATCCTTGCAGGACCAAGGAGACTATGTCTGCCTTGCTCAAGACAGGAAGACCAAGAAAAGACATTGCGTGGTCAGG CAGCTCACAGTCCTAGAGCGTTAA.


11. The expression vector of claim 8 wherein the DNA encoding thesVEGF-RTMI comprises the nucleotide sequence: (SEQ. ID. NO.: 17)GCGCTCACCATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTGAACTGAGTTTAAAAGGCACCCAGCACATCATGCAAGCAGGCCAGACACTGCATCTCCAATGCAGGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGTGAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCCTGTGGAAGAAATGGCAAACAATTCTGCAGTACTTTAACCTTGAACACAGCTCAAGCAAACCACACTGGCTTCTACAGCTGCAAATATCTAGCTGTACCTACTTCAAAGAAGAAGGAAACAGAATCTGCAATCTATATATTTATTAGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCAATACAATCATAGATGTCCAAATAAGCACACCACGCCCAGTCAAATTACTTAGAGGCCATACTCTTGTCCTCAATTGTACTGCTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAGTTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGACGAATTGACCAAAGCAATTCCCATGCCAACATATTCTACAGTGTTCTTACTATTGACAAAATGCAGAACAAAGACAAAGGACTTTATACTTGTCGTGTAAGGAGTGGACCATCATTCAAATCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCATCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGTAGCTGGCAAGCGGTCTTACCGGCTCTCTATGAAAGTGAAGGCATTTCCCTCGCCGGAAGTTGTATGGTTAAAAGATGGGTTACCTGCGACTGAGAAATCTGCTCGCTATTTGACTCGTGGCTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCAGGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATGTGTTTAAAAACCTCACTGCCACTCTAATTGTCAATGTGAAACCCCAGATTTACGAAAAGGCCGTGTCATCGTTTCCAGACCCGGCTCTCTACCCACTGGGCAGCAGACAAATCCTGACTTGTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTTCTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGTGACTTTTGTTCCAATAATGAAGAGTCCTTTATCCTGGATGCTGACAGCAACATGGGAAACAGAATTGAGAGCATCACTCAGCGCATGGCAATAATAGAAGGAAAGAATAAGATGGCTAGCACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACATTTGCATAGCTTCCAATAAAGTTGGGACTGTGGGAAGAAACATAAGCTTTTATATCACAGATGTGCCAAATGGGTTTCATGTTAACTTGGAAAAAATGCCGACGGAAGGAGAGGACCTGAAACTGTCTTGCACAGTTAACAAGTTCTTATACAGAGACGTTACTTGGATTTTACTGCGGACAGTTAATAACAGAACAATGCACTACAGTATTAGCAAGCAAAAAATGGCCATCACTAAGGAGCACTCCATCACTCTTAATCTTACCATCATGAATGTTTCCCTGCAAGATTCAGGCACCTATGCCTGCAGAGCCAGGAATGTATACACAGGGGAAGAAATCCTCCAGAAGAAAGAAATTACAATCAGAGATCAGGAAGCACCATACCTCCTGCGAAACCTCAGTGATCACACAGTGGCCATCAGCAGTTCCACCACTTTAGACTGTCATGCTAATGGTGTCCCCGAGCCTCAGATCACTTGGTTTAAAAACAACCACAAAATACAACAAGAGCCTGGAATTATTTTAGGACCAGGAAGCAGCACGCTGTTTATTGAAAGAGTCACAGAAGAGGATGAAGGTGTCTATCACTGCAAAGCCACCAACCAGAAGGGCTCTGTGGAAAGTTCAGCATACCTCACTGTTCAAGGAACCTCGGACAAGTCTAATCTGGAGCTGATCACTCTAACATGCACCTGTGTGGCTGCGACTCTCTTCTGGC TCCTATTAACCCTCCTTATCTAA


12. The expression vector of claim 8 wherein the DNA encoding thesVEGF-RTMII comprises the nucleotide sequence: (SEQ. ID. NO.: 18)CTCGAGGTGCAGGATGCAGAGCAAGGTGCTGCTGGCCGTCGCCCTGTGGCTCTGCGTGGAGACCCGGGCCGCCTCTGTGGGTTTGCCTAGTGTTTCTCTTGATCTGCCCAGGCTCAGCATACAAAAAGACATACTTACAATTAAGGCTAATACAACTCTTCAAATTACTTGCAGGGGACAGAGGGACTTGGACTGGCTTTGGCCCAATAATCAGAGTGGCAGTGAGCAAAGGGTGGAGGTGACTGAGTGCAGCGATGGCCTCTTCTGTAAGACACTCACAATTCCAAAAGTGATCGGAAATGACACTGGAGCCTACAAGTGCTTCTACCGGGAAACTGACTTGGCCTCGGTCATTTATGTCTATGTTCAAGATTACAGATCTCCATTTATTGCTTCTGTTAGTGACCAACATGGAGTCGTGTACATTACTGAGAACAAAAACAAAACTGTGGTGATTCCATGTCTCGGGTCCATTTCAAATCTCAACGTGTCACTTTGTGCAAGATACCCAGAAAAGAGATTTGTTCCTGATGGTAACAGAATTTCCTGGGACAGCAAGAAGGGCTTTACTATTCCCAGCTACATGATCAGCTATGCTGGCATGGTCTTCTGTGAAGCAAAAATTAATGATGAAAGTTACCAGTCTATTATGTACATAGTTGTCGTTGTAGGGTATAGGATTTATGATGTGGTTCTGAGTCCGTCTCATGGAATTGAACTATCTGTTGGAGAAAAGCTTGTCTTAAATTGTACAGCAAGAACTGAACTAAATGTGGGGATTGACTTCAACTGGGAATACCCTTCTTCGAAGCATCAGCATAAGAAACTTGTAAACCGAGACCTAAAAACCCAGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGTGTAACCCGGAGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTGGGCTGATGACCAAGAAGAACAGCACATTTGTCAGGGTCCATGAAAAACCTTTTGTTGCTTTTGGAAGTGGCATGGAATCTCTGGTGGAAGCCACGGTGGGGGAGCGTGTCAGAATCCCTGCGAAGTACCTTGGTTACCCACCCCCAGAAATAAAATGGTATAAAAATGGAATACCCCTTGAGTCCAATCACACAATTAAAGCGGGGCATGTACTGACGATTATGGAAGTGAGTGAAAGAGACACAGGAAATTACACTGTCATCCTTACCAATCCCATTTCAAAGGAGAAGCAGAGCCATGTGGTCTCTCTGGTTGTGTATGTCCCACCCCAGATTGGTGAGAAATCTCTAATCTCTCCTGTGGATTCCTACCAGTACGGCACCACTCAAACGCTGACATGTACGGTCTATGCCATTCCTCCCCCGCATCACATCCACTGGTATTGGCAGTTGGAGGAAGAGTGCGCCAACGAGCCCAGCCAAGCTGTCTCAGTGACAAACCCATACCCTTGTGAAGAATGGAGAAGTGTGGAGGACTTCCAGGGAGGAAATAAAATTGCCGTTAATAAAAATCAATTTGCTCTAATTGAAGGAAAAAACAAAACTGTAAGTACCCTTGTTATCCAAGCGGCAAATGTGTCAGCTTTGTACAAATGTGAAGCGGTCAACAAAGTCGGGAGAGGAGAGAGGGTGATCTCCTTCCACGTGACCAGGGGTCCTGAAATTACTTTGCAACCTGACATGCAGCCCACTGAGCAGGAGAGCGTGTCTTTGTGGTGCACTGCAGACAGATCTACGTTTGAGAACCTCACATGGTACAAGCTTGGCCCACAGCCTCTGCCAATCCATGTGGGAGAGTTGCCCACACCTGTTTGCAAGAACTTGGATACTCTTTGGAAATTGAATGCCACCATGTTCTCTAATAGCACAAATGACATTTTGATCATGGAGCTTAAGAATGCATCCTTGCAGGACCAAGGAGACTATGTCTGCCTTGCTCAAGACAGGAAGACCAAGAAAAGACATTGCGTGGTCAGGCAGCTCACAGTCCTAGAGCGTGTGGCACCCACGATCACAGGAAACCTGGAGAATCAGACGACAAGTATTGGGGAAAGCATCGAAGTCTCATGCACGGCATCTGGGAATCCCCCTCCACAGATCATGTGGTTTAAAGATAATGAGACCCTTGTAGAAGACTCAGGCATTGTATTGAAGGATGGGAACCGGAACCTCACTATCCGCAGAGTGAGGAAGGAGGACGAAGGCCTCTACACCTGCCAGGCATGCAGTGTTCTTGGCTGTGCAAAAGTGGAGGCATTTTTCATAATAGAAGGTGCCCAGGAAAAGACGAACTTGGAAATCATTATTCTAGTAGGCACGACGGTGATTGCCATGTTCTTCTGGCTACTTCTTGTCATCATCCT AGGGACCGTTTAA.


13. A recombinant host cell containing the expression vector of claim 8.14. A method for inhibiting VEGF receptor function comprising theadministration of the VEGF inhibitor of claim 1 in an amount sufficientto inhibit VEGF receptor function.
 15. The method of claim 14 whereinthe VEGF inhibitor is selected from the group consisting of sVEGF-RI,sVEGF-RII, sVEGF-RTMI, and sVEGF-RTMII.
 16. A pharmaceutical compositioncomprising the inhibitor of claim 1 and a pharmaceutically acceptablecarrier.
 17. The pharmaceutical composition of claim 16 wherein theinhibitor is selected from the group consisting of sVEGF-RI, sVEGF-MI,sVEGF-RTMI, and sVEGF-RTMII.
 18. A method for inhibiting angiogenesiscomprising the administration of the VEGF inhibitor of claim 1 in anamount sufficient to inhibit angiogensis.